The laboratory is interested in understanding the clathrin-independent endocytosis pathway that is associated with Arf6. We have shown that this pathway is responsible for internalizing plasma membrane (PM) proteins that lack sequences that allow recognition by the clathrin and adaptor protein machinery. Among the proteins that enter cells via this mode of endocytosis are the major histocompatibility complex Class I protein (MHCI) and the lipid anchored protein CD59. After endocytosis, MHCI and CD59 in vesicles are delivered to and fuse with endosomes containing cargo proteins from the clathrin-dependent endocytosis pathway such as transferrin receptor. From there, MHCI and CD59 can proceed on to late endosomal compartments where they are degraded or they can be recycled back out to the PM via unique tubular recycling endosomes. In HeLa cells these recycling endosomes contain only cargo that had entered via clathrin-independent endocytosis and their return to the PM is dependent upon the activity of Arf6 and several other regulators including Rab11 and 22. In an attempt to discover the machinery and additional cargo proteins associated with this pathway, we have isolated clathrin-independent endosomes and used mass spectrometry to identify new candidate proteins. We have validated 7 new cargo proteins that enter cells and traffic along this clathrin-independent pathway: CD44, CD55, CD98, CD147, Lat1, ICAM1 and the non-insulin stimulated glucose transporter Glut1 (Eyster et al 2009). We have confirmed that these proteins enter cells by clathrin-independent endocytosis in many human cell lines and in addition, in HeLa cells, we see a difference in intracellular itinerary followed by these proteins. CD55 and Glut1 follow a similar itinerary as MHCI, merging with endosomes containing the transferrin receptor before routing to the tubular recycling endosomes. By contrast, CD44, 98 and 147 after endocytosis do not enter transferrin-containing endosomes but are routed directly to the recycling endosome (Eyster et al 2009). This divergence in itinerary suggests that clathrin-independent cargo proteins are sorted within the cell and we are currently studying whether signals contained on these cargo proteins (extracellular, transmembrane or cytoplasmic) are directing their trafficking. In another project in the lab, we investigated whether this clathrin-independent endocytic pathway might be utilized for constitutive trafficking of signaling receptors. G protein-coupled receptors (GPCRs) are a large family of seven transmembrane-spanning proteins that respond to many cellular signals or ligands. In most cases, following ligand binding the GPCRs are rapidly internalized into cells by clathrin-dependent endocytosis. We found that both the b2 adrenergic and M3 muscarinic receptors entered cells via clathrin-independent endocytosis in the absence of ligand and traveled along the same recycling pathway as MHCI (Scarselli and Donaldson, 2009). Furthermore, the endogenous G proteins that are activated by these receptors, alpha s and alpha q, were localized also to these membrane systems. In the presence of ligand, now the GPCR entered cells via clathrin-dependent endocytosis, but the G proteins remained at the cell surface and associated with clathrin-independent endosomes. Interestingly, deletion of the third intracellular loop of the M3 receptor results in a receptor capable of full signaling but unable to enter cells via clathrin-mediated endocytosis. This truncated M3 receptor was still rapidly internalized upon exposure to ligand, but now entered cells via clathrin-independent endocytosis (Scarselli and Donaldson, 2009). This demonstrates that GPCRs represent cell surface proteins that can enter different types of endocytic pathways depending on whether the receptor is stimulated with ligand or when ligand is absent where it might undergo constitutive endocytic trafficking. In addition to regulating recycling of endosomal membrane back to the cell surface, Arf6 has also been implicated in some forms of regulated secretion. Both of these processes generally require the actin cytoskeleton for exocytosis of the membrane back to the plasma membrane. In a collaborative study, it was found that myosin Vc is associated with tubular endosomes in breast cancer cell line MCF7 and also with secretory granules (Jacobs et al 2009). Although it is not clear whether myosin Vc is associated with the recycling endosomes in HeLa cells, we are investigating roles for other motor proteins, including microtubular-based motors, in this process